Aspergillus terreus is the most important strain for the industrial production of itaconic acid. Few works have reported the critical metabolic nodes in the synthesis of itaconic acid in A. terreus. Herein a high-throughput screening method was established to screen an efficient mutant A. terreus 6–12#, which could produce 28.6 g/L itaconic acid with a productivity of 0.36 g/g glucose, about 66 % and 57 % higher than that of the parent strain. On this basis, the expression of the key enzymes was analyzed centering on the glycolytic pathway, the precursor synthesis pathway, the itaconic acid synthesis and degradation pathways, and the membrane transport system. It was proven that the expressions of enzymes favoring itaconic acid synthesis in each of these modules were significantly elevated in the mutant. But the enzymes CAD, MTT, MFS in the itaconic acid synthesis pathway and the membrane transport system displayed low elevation levels, while the degradation pathway was activated with the improvement of itaconic acid, which was considered the possible limited step in itaconic acid synthesis in A. terreus. These findings provided potential metabolic modification targets for further acquisition of excellent A. terreus cell factories and helped to facilitate the industrial production of itaconic acid.